Artificial insemination (AI) has been used in dairy cattle and pigs for several decades. However, methods of improving sperm quality in sperm doses used for AI are required for several reasons: (i) to remove spermatozoa from inhibiting factors in seminal plasma, e.g. decapacitating factors; (ii) to select the mature, normal and viable spermatozoa from the entire sperm population in the ejaculate; (iii) to separate spermatozoa from sources of reactive oxygen species which are detrimental to sperm survival. These functions are normally carried out by the female reproductive tract during natural mating but may be partly lacking when spermatozoa are artificially inseminated. Furthermore, when spermatozoa are to be used for in vitro fertilisation, these selection mechanisms are completely absent and the spermatozoa must be separated from the seminal plasma prior to use.
Artificial insemination (AI) has been used in dairy cattle and pigs for several decades. However, methods of improving sperm quality in sperm doses used for AI are required for several reasons: (i) to remove spermatozoa from inhibiting factors in seminal plasma, e.g. decapacitating factors; (ii) to select the mature, normal and viable spermatozoa from the entire sperm population in the ejaculate; (iii) to separate spermatozoa from sources of reactive oxygen species which are detrimental to sperm survival. These functions are normally carried out by the female reproductive tract during natural mating but may be partly lacking when spermatozoa are artificially inseminated. Furthermore, when spermatozoa are to be used for in vitro fertilisation, these selection mechanisms are completely absent and the spermatozoa must be separated from the seminal plasma prior to use.
Over the last 15 years, the technique of density gradient centrifugation has been used to prepare human spermatozoa for use in assisted reproduction (WHO, 1999). Originally, silica particles coated with polyvinylpyrrolidone were used, but more recent formulations have used silane-coated silica particles in the density gradient.
The prepared sperm suspensions are used immediately for in vitro fertilization, intra-cytoplasmic sperm injection or intrauterine deposition.
Commercially available colloid formulations for preparing human spermatozoa e.g. Puresperm, (Nidacon International AB) for assisted reproduction have an osmolarity of 300-310 mOsm, according to the company website and promotional literature. Until now, the only commercially available density gradient products for animal spermatozoa quoted in the literature also have an osmolarity within the range 300-310 mOsm, eg. Equipure and Bovipure (both made by Nidacon International). Although Bovipure was reported to give good results when used to prepare bovine spermatozoa for IVF (4), the use of Equipure as a density gradient for stallion spermatozoa did not give the same beneficial effects on sperm quality (5) as have been reported for human spermatozoa (6).
Preparation of human semen samples is commonly performed in aliquots of 1.5 mL semen. Although preparation of animal semen in such small volumes is adequate for preparing spermatozoa for IVF or ICSI, much larger sperm numbers are needed for artificial insemination in animals. Consequently, there is a need for simple and convenient methods for preparation of spermatozoa from animal semen at the site of collection and in adequate amounts for use in artificial insemination. There is also a need for animal-specific compositions suitable for use in such methods.